Methods for treating conditions associated with the accumulation of excess extracellular matrix

ABSTRACT

The present invention is methods and compositions for reducing and preventing the excess accumulation of extracellular matrix in a tissue and/or organ or at a wound site using a combination of agents that inhibit TGFβ, or using agents that inhibit TGFβ in combination with agents that degrade excess accumulated extracellular matrix. The compositions and methods of the invention are used to treat conditions such as fibrotic diseases and scarring that result from excess accumulation of extracellular matrix, impairing tissue or organ function or skin appearance in a subject.

This application claims the priority of International Application No.PCT/US00/0179, filed Jan. 5, 2000, which claims the priority of U.S.Ser. No. 60/114,795, filed Jan. 5, 1999.

This invention was made with government support under Grant No.5R01DK49374 and 2R37DK043609 awarded by the National Institute ofHealth. The government has certain rights to this invention.

FIELD OF THE INVENTION

This invention relates to a method for preventing or reducing excessaccumulation of extracellular matrix in tissues or organs or at a woundsite, and more particularly to the prevention and treatment ofconditions resulting from excess accumulation of extracellular matrix,using a combination of agents that inhibit TGFβ, or a combination ofagents that inhibit TGFβ and agents that degrade excess accumulatedextracellular matrix.

BACKGROUND OF THE INVENTION

Excess deposition and accumulation of extracellular matrix (ECM) isfound in diseases such as fibrosis of the kidney or lung. Although thecytokine transforming growth factor Beta (TGFβ) regulates extracellularmatrix deposition for tissue repair, overproduction of TGFβ clearlyunderlies tissue fibrosis caused by excess deposition of extracellularmatrix resulting in disease (Border and Ruoslahti, J. Clin. Invest.90:1-7 (1992)). TGFβ's fibrogenic action results from simultaneousstimulation of matrix protein synthesis (Border et al., Kidney Int37:689-695 (1990), inhibition of matrix degradation and turnover andenhanced cell-matrix interactions through modulation of integrinreceptors that facilitate ECM assembly. Overproduction of TGFβ has beendemonstrated in glomerulonephritis (Okuda et al., J. Clin. Invest.86:453-462 (1990)), diabetic nephropathy and hypertensive glomerularinjury and in related fibrotic disorders of the lung, liver, heart,arterial wall, skin, brain, joints and bone marrow (Border and Noble, N.Eng. J. Med. 331:128-1292 (1994)). In addition to the kidney, blockingthe action of TGFβ with an agent such as antibody or the proteoglycandecorin has been shown to be therapeutic in fibrosis and scarring of theskin, lung, central nervous system and arterial wall (Border and Noble,Kidney Int. 51:1388-1396 (1997)).

Suppression of the production of ECM and prevention of excessaccumulation of mesangial matrix in glomeruli of glomerulonephritic ratshas been demonstrated by intravenous administration of neutralizingantibodies specific for TGFβ (Border et al., Nature 346:371-374 (1990))or administration of purified decorin, a proteoglycan (Border et al.,Nature 360:361-364 (1992)) and by introduction of nucleic acid encodingdecorin, a TGFβ-inhibitory agent, into a rat model of acute mesangialglomerulonephritis (Isaka et al., Nature Med. 2:418-423 (1996)).Inhibition of TGFβ activity, using for example anti-TGFβ antibodies, hasbeen shown to to disrupt TGFβ overproduction (Sharma et al., Diabetes45:522-530 (1996)).

Dermal scarring following dermal injury results from excessiveaccumulation of fibrous tissue made up of collagen, fibronectin andproteoglycans at a wound site. Because the fibrous extracellular matrixlacks elasticity, scar tissue can impair essential tissue function aswell as result in an undesirable cosmetic appearance. TGFβ is believedto induce the deposition of fibrous matrix at the wound site (Shah etal., Lancet 339:213-214 (1992)).

One explanation for persistent TGFβ overexpression in progressivefibrotic kidney disease is that repeated or multiple episodes of tissueinjury, such as occurs in chronic diseases such as hypertension,diabetes or immune complex disease lead to continuous overproduction ofTGFβ and extracellular matrix resulting in tissue fibrosis (See Borderand Noble, N. Eng. J. Med. 331:1286-1292(1994)). Another possibleexplanation for persistent TGFα overexpression is the presence of abiologically complex interconnection between TGFβ and therenin-angiotensin system (RAS) in the kidney as part of an emergencysystem that responds to the threat of tissue injury as discussed furtherherein.

Renin is an aspartyl proteinase synthesized by juxtaglomerular kidneycells and mesangial cells in humans and rats. (Chansel et al., Am. J.Physiol. 252:F32-F38 (1987) and Dzau and Kreisberg, J. Cardiovasc.Pharmacol. 8(Suppl 10):S6-S10(1986)). Renin plays a key role in theregulation of blood pressure and salt balance. Its major source inhumans is the kidney where it is initially produced as preprorenin.Signal peptide processing and glycosylation are followed by secretion ofprorenin and its enzymatically active form, mature renin. The activeenzyme triggers a proteolytic cascade by cleaving angiotensinogen togenerate angiotensin I, which is in turn converted to the vasoactivehormone angiotensin II by angiotensin converting enzyme (“ACE”).

The sequence of the human renin gene is known (GenBank entry M2690).Recombinant human renin has been synthesized and expressed in variousexpression systems (Sielecki et al., Science 243:1346-1351(1988),Mathews et al., Protein Expression and Purification 7:81-91 (1996)).Inhibitors of renin's enzymatic site are known (Rahuel et al., J.Struct. Biol. 107:227-236 (1991); Badasso et al., J. Mol. Biol.223:447-453 (1992); and Dhanaraj et al., Nature 357:466-472 (1992))including an orally active renin inhibitor in primates, Ro 42-5892(Fischli et al., Hypertension 18:22-31(1991)). Renin-binding proteinsand a cell surface renin receptor on human mesangial cells have beenidentified (Campbell and Valentijn, J. Hypertens. 12:879-890 (1994),Nguyen et al., Kidney Internat. 50:1897-1903 (1996) and Sealey et al.,Amer. J. Hyper. 9:491-502 (1996)).

The renin-angiotensin system (RAS) is a prototypical systemic endocrinenetwork whose actions in the kidney and adrenal glands regulate bloodpressure, intravascular volume and electrolyte balance. In contrast,TGFβ is considered to be a prototypical cytokine, a peptide signalingmolecule whose multiple actions on cells are mediated in a local orparacrine manner. Recent data however, indicate that there is an intactRAS in many tissues whose actions are entirely paracrine and TGFα haswide-ranging systemic (endocrine) effects. Moreover, RAS and TGFε act atvarious points to regulate the actions of one another.

In a systemic response to an injury such as a wound, the RAS rapidlygenerates AII that acts by vasoconstriction to maintain blood pressureand later stimulates the secretion of aldosterone, resulting in anincrease in intravascular volume. In the wound, TGFβ is rapidly releasedby degranulating platelets and causes a number of effects including: 1)autoinduction of the production of TGFβ by local cells to amplifybiological effects; 2) chemoattraction of monocyte/macrophages thatdebride and sterilize the wound and fibroblasts that begin synthesis ofECM; 3) causing deposition of new ECM by simultaneously stimulating thesynthesis of new ECM, inhibiting the proteases that degrade matrix andmodulating the numbers of integrin receptors to facilitate cell adhesionto the newly assembled matrix; 4) suppressing the proinflammatoryeffects of interleukin-1 and tumor necrosis factor; 5) regulating theaction of platelet derived growth factor and fibroblast growth factor sothat cell proliferation and angiogenesis are coordinated with matrixdeposition; and 6) terminating the process when repair is complete andthe wound is closed (Border and Noble, Scientific Amer. Sci. & Med.2:68-77 (1995)).

Interactions between RAS and TGFβ occur at both the systemic andmolecular level. It has been shown that TGFβ's action in causing ECMdeposition in a healing wound is the same action that makes TGFβ apowerful fibrogenic cytokine. (Border and Noble, New Engl. J. Med.331:1286-1292(1994); and Border and Ruoslahti, J. Clin. Invest. 90:107(1992). Indeed, it is the failure to terminate the production of TGFβthat distinguishes normal tissue repair from fibrotic disease. RAS andTGFβ co-regulate each other's expression. Thus, both systems may remainactive long after an emergency response has been terminated, which canlead to progressive fibrosis. The kidney is particularly susceptible tooverexpression of TGFβ. The interrelationship of RAS and TGFβ mayexplain the susceptibility of the kidney to TGFβ overexpression and whypharmacologic suppression of RAS or inhibition of TGFβ are boththerapeutic in fibrotic diseases of the kidney. (Noble and Border, Sem.Nephrol., supra and Border and Noble, Kidney Int. 51:1388-1396 (1997)).

Activation of RAS and generation of angiotensin II (AII) are known toplay a role in the pathogenesis of hypertension and renal and cardiacfibrosis. TGFβ has been shown to be a powerful fibrogenic cytokine,acting simultaneously to stimulate the synthesis of ECM, inhibit theaction of proteases that degrade ECM and increasing the expression ofcell surface integrins that interact with matrix components. Throughthese effects, TGFβ rapidly causes the deposition of excess ECM. AIIinfusion strongly stimulates the production and activation of TGFβ inthe kidney. (Kagami et al., J. Clin. Invest. 93:2431-2437 (1994)).Angiotensin II also upregulates TGFβ production and increases activationwhen added to cultured vascular smooth muscle cells (Gibbons et al, J.Clin. Invest. 90:456-461 (1992)) and this increase is independent ofpressure (Kagami et al., supra). AII also upregulates TGFβ receptors,even in the presence of exogenously added TGFβ which normallydown-regulates its own receptors, leading to enhanced TGFβ signallingand enhanced fibronectin production (Kanai et al., J. Am. Soc. Nephrol.8:518A (1997)). Blockade of AII reduces TGFβ overexpression in kidneyand heart, and it is thought that TGFβ mediates renal and cardiacfibrosis associated with activation of RAS (Noble and Border, Sem.Nephrol. 17(5):455-466 (1997)), Peters et al., Kidney International 54(1998)). Blockade of AII using inhibitors of ACE slow the progression ofrenal fibrotic disease (see, e.g., Anderson et al., J. Clin. Invest.76:612-619 (1985) and Noble and Border, Sem. Nephrol. 17(5):455-466(1997)). What is not clear is whether angiotensin blockade reducesfibrosis solely through controlling glomerular hypertension and therebyglomerular injury, or whether pressure-independent as well aspressure-dependent mechanisms are operating. While ACE inhibitors andAII receptor antagonists have been shown to slow the progress offibrotic diseases, they do not halt disease and TGFβ levels remainsomewhat elevated. (Peters et al., supra).

Thus, RAS and TGFβ can be viewed as powerful effector molecules thatinteract to preserve systemic and tissue homeostasis. The response to anemergency such as tissue injury is that RAS and TGFβ become activated.Continued activation may result in chronic hypertension and progressivetissue fibrosis leading to organ failure. Because of the interplaybetween the RAS and TGFβ, and the effects of this interplay on tissuehomeostasis, blockade of the RAS may be suboptimal to prevent or treatprogressive fibrotic diseases such as diabetic nephropathy.

Components of the renin-angiotensin system act to further stimulateproduction of TGFβ and plasminogen activator inhibitor leading to rapidECM accumulation. The protective effect of inhibition of therenin-angiotensin system in experimental and human kidney diseasescorrelates with the suppression of TGFβ production. (Noble and Border,Sem. Nephrol., supra; and Peters et al., supra).

The renin molecule has been shown to enzymatically cleaveangiotensinogen into angiotensin I. The angiotensin I is then convertedby Angiotensin Converting Enzyme (“ACE”) to Angiotensin II which acts asan active metabolite and induces TGFβ production. Angiotensin II is animportant modulator of systemic blood pressure. It has been thought thatif you decrease hypertension by blocking AII's vasoconstrictor effectsfibrotic disease is reduced.

In the glomerular endothelium, activation of RAS and TGFβ have beenshown to play a role in the pathogenesis of glomerulonephritis andhypertensive injury. Volume (water) depletion and restriction ofpotassium have been shown to stimulate both production of renin and TGFβin the juxtaglomerular apparatus (JGA) of the kidney (Horikoshi et al.,J. Clin. Invest. 88:2117-2122 (1992) and Ray et al., Kidney Int.44:1006-1013 (1993)). Angiotensin blockade has also been shown toincrease the production of renin. TGFβ has been shown to stimulate therelease of renin from kidney cortical slices and cultured JG cells(Antonipillai et al., Am. J. Physiol. 265:F537-F541 (1993); Ray et al.,Contrib. Nephrol. 118:238-248 (1996) and Veniant et al., J. Clin.Invest. 98:1996-19970 (1996)), suggesting that renin and TGFβ arecoregulated. Other interactions between RAS and TGFβ include that AIIinduces the production of TGFβ in cultured cells and in vivo (Kagami etal., supra) and AII regulates expression of TGFβ receptors (Kanai etal., 1977, supra). It is thus likely that the fibrogenic effects thathave been attributed to AII are actually mediated by TGF.

Another interplay between RAS and TGFβ is with the production ofaldosterone. Aldosterone overproduction has been linked to hypertensionand glomerulosclerosis. AII stimulates the production and release ofaldosterone from the adrenal gland. In contrast, TGFβ suppressesaldosterone production and blocks the ability of AII to stimulatealdosterone by reducing the number of AII receptors expressed in theadrenal (Gupta et al., Endocrinol. 131:631-636 (1992)), and blocks theeffects of aldosterone on sodium reabsorption in cultured renalcollecting duct cells (Husted et al., Am. J. Physiol. Renal, FluidElectrolyte Physiol. 267:F767-F775 (1994)). Aldosterone may havefibrogenic effects independent of AII, and may upregulate TGFβexpression. The mechanism of aldosterone's pathological effects isunknown but might be due to stimulation of TGFβ production in the kidney(Greene et al., J. Clin. Invest. 98:1063-1068 (1996)).

Prorenin or renin may have AII-independent actions to increase fibroticdisease. Prorenin overexpressing rats were found to be normotensive butto develop severe glomerulosclerosis (Veniant et al., J. Clin. Invest.98:1996-1970 (1996)).

Human recombinant renin added to human mesangial cells induces markedupregulation of production of plasminogen activator inhibitors (e.g.PAI-1 and PAI-2) which block the generation of plasmin, a fibrinolyticenzyme important in the dissolution of clots after wounding generatedfrom plasminogen by two enzymes called plasminogen activators, urokinase(u-PA) and tissue plasminogen activator (t-PA). PAI-1 and 2 regulateU-PA and t-PA in turn. Plasmin appears to be a key mediator ofextracellular matrix degradation, carrying out at least three functionsimportant to extracellular matrix degradation. Plasmin directly degradesproteoglycan components of extracellular matrix, proteolyticallyactivates metalloproteinases (MMPs) that, in turn, degrade collagens andother matrix proteins, and enzymatically inactivates tissue inhibitorsof MMPs (TIMPs), releasing MMPs from inhibition of TIMPs, allowing themto proteolytically digest matrix proteins. (Baricos et al., KidneyInt'l. 47:1039-1047(1995); Baricos et al., J. Amer. Soc. Nephrol.10:790-795 (1999)). The net generation of active plasmin from theinactive precursor plasminogen results from a balance of the plasminogenactivators and PAI-1 and 2, and other factors. PAI-1 binds tovitronectin. (Lawrence et al., J. Biol. Chem. 272:7676-7680 (1997)).Mutant PAI-1 molecules have been developed that have enhanced propertiesfor PAI-1 binding to vitronectin molecules, but do not inhibit eithert-PA or u-PA activity, resulting in an increase in the amount of theactive form of plasmin. (See, WO 97/39028, Lawrence et al.). PAI-1 isincreased in response to added TGFβ (Tomooka et al., Kidney Int.42:1462-1469 (1992)).

It has been suggested that TGFβ enhances release of renin from storagegranules in the juxtaglomerular apparatus of the kidney (Antonipillai etal., Am. J. Physiol. 265:F537-F541 (1993) and Ray et al., Contrib.Nephrol. 118:238-248 (1996)).

Thus, the interactions of RAS and TGFβ production form a complex systemwhich impacts fibrotic ECM accumulation and the incidence of fibroticdisease. Various RAS components such as aldosterone, prorenin and reninmay be connected with TGFβ production and fibrotic ECM accumulation. Anysuccessful therapeutic regime must take into account these complexrelationships to optimize inhibition of TGFβ to prevent and/or reduceECM accumulation.

The multiple pathways resulting in TGFβ overexpression and fibrosisproposed from in vitro studies are depicted in FIG. 1. (See, Kagami etal., J. Clin. Invest. 93:2431-2437 (1994); Gibbons et al., J. Clin.Invest. 90:456-461 (1992); Abboud, Kidney Int. 41:581-583 (1992);Ruiz-Ortega et al., J. Am. Soc. Nephrol. 5:683 (1994) abstract; Kim etal., J. Biol. Chem. 267:13702-13707 (1992); Ohno et al., J. Clin.Invest. 95:1363-1369 (1995); Riser et al, J. Clin. Invest. 90:1932-1943(1992); Riser et al., J. Am. Soc. Nephrol. 4:663 (1993); Ziyadeh et al.,J. Clin. Invest. 93:536-542 (1994); Rocco et al., Kidney Int. 41:107-114(1992); Flaumenhaft et al., Advan. Pharmacol. 24:51-76 (1993);Lopez-Armanda et al., J. Am. Soc. Nepbrol. 5:812 (1994) abstract; Sahaiet al., J. Am. Soc. Nephrol. 6:910 (1995); Remuzzi et al., Kidney Int.1:2-15 (1997); and Remuzzi et al., J. Am. Soc. Nephrol. 9:1321-1332(1998)). This diagram shows that a large number of factors implicated inkidney injury are believed to increase the production of TGFβ.

In fibrotic diseases overproduction of TGFβ results in excessaccumulation of extracellular matrix which leads to tissue fibrosis andeventually organ failure. Accumulation of mesangial matrix is ahistological indication of progressive glomerular diseases that lead toglomerulosclerosis and end-stage kidney disease (Klahr et al., N. Engl.J. Med. 318:1657-1666 (1988); Kashgarian and Sterzel, Kidney Int.41:524-529 (1992)). Rats injected with antithymocyte serum are anaccepted model of human glomerulonephritis and this model hasdemonstrated that overproduction of glomerular TGFβ can underlie thedevelopment of glomerulosclerosis (Okuda et al., J. Clin. Invest.86:453-462 (1990); Border et al., Nature (Lond.) 346:371-374 (1990);Kagami et al., Lab. Invest. 69:68-76 (1993); and Isaka et al., J. Clin.Invest. 92:2597-2602 (1993)). Using cultured rat mesangial cells wherethe effects of Angiotensin II on glomerular pressure are not a factor,Angiotensin II has been shown to induce TGFβ production and secretion bymesangial cells, and this in turn has been shown to stimulateextracellular matrix production and deposition (Kagami et al., J. Clin.Invest. 93:2431-2437 (1994)). Increases in PAI-1 levels result indecreased degradation of extracellular matrix (Baricos et al., KidneyInt. 47:1039-1047 (1995)). Increases in TGFβ result in increased PAI-1levels (Tomooka et al., Kidney Int. 42:1462-1469 (1992)). It has beendemonstrated that decreasing TGFβ overexpression in a rat model ofglomerulonephritis by in vivo injection of neutralizing antibodies toTGFβ, reduces TGFβ overexpression (Border et al., Nature 346:371-374(1990)), and reduces PAI-1 deposition into the pathological matrix(Tomooka et al., Kidney Int. 42:1462-1469 (1992)). Therefore, decreasesin TGFβ levels should result in decreased PAI-1 levels and increaseddegradation of extracellular matrix to ameliorate organ impairment andfibrotic disease. However, patients present with fibrotic disease thatis well advanced in terms of build-up of extra-cellular matrix (ECM).This is because abnormal organ function is undetectable until ECMaccumulation is very advanced. For example, in the kidney, standarddiagnostic tests do not provide an abnormal reading until about fiftypercent of organ function has been lost.

The treatment of conditions associated with excess accumulation of ECMhas also focused on decreasing stimuli to disease such as to lower bloodpressure or, in the case of diabetic nephropathy to reduce plasmaglucose levels. For example, current therapies for treating fibroticdisease in the kidney are limited to AII blockade using ACE inhibitorssuch as Enalapril or AII receptor antagonists such as Losartan. Inaddition, patients are encouraged to follow low protein diets since thisregimen has some therapeutic value (Rosenberg et al., J. Clin. Invest.85:1144-1149(1992)). These therapies, at best, prolong organ function byonly 1-2 years. This may be because of the multiple pathways that resultin TGFβ overexpression or enhanced activity. Moreover, it is likely thatcurrent therapeutic strategies to reduce TGFβ overproduction may lead toupregulation of other pathways resulting in continued TGFβoverproduction. For example, when the action of AII is blocked, renin isupregulated which itself increases TGFβ production (see co-pending U.S.patent application, U.S. Ser. No. 09/005,255, incorporated in itsentirety herein). More recently, treatments aimed to halt theoverproduction of TGFβ have been proposed (Border and Noble, KidneyInternatl. 54 (1998); and Peters et al., Kidney Internatl. 54 (1998)).

Therefore, the most promising therapeutic methods will need to increaseECM degradation to restore organ function as well as decrease TGFβoverproduction and/or activity. Enhanced degradation of excessaccumulated ECM can be used to optimize overall reduction in levels ofaccumulated ECM to restore function to tissues and organs. Proteasesthat are able to degrade ECM are known. For example, the serine proteaseplasmin degrades ECM proteins and activates pro-metalloproteinases, inaddition to degrading fibrin (Baricos et al., supra). One goal oftherapeutic intervention to increase ECM degradation for treatingfibrosis could be increasing plasmin in the region of excess ECMdeposition.

There is a need for improved therapies to normalize TGFβ production,that take into account the multiple pathways that stimulate TGFβproduction, to prevent or reduce excess accumulation of ECM, to restorefunction to tissues and organs in which excess ECM has accumulatedand/or to reduce scar formation at a wound site.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides methods for preventing orreducing the excess accumulation of extracellular matrix (ECM)associated with fibrotic conditions by inhibiting TGFβ, using acombination of agents that inhibit TGFβ, or by using a combination ofagents to inhibit TGFβ and agents that cause the enhanced degradation ofexcess accumulated ECM.

The methods of the invention contemplate the use of agents that directlyor indirectly inhibit TGFβ including direct inhibitors of TGFβ activitysuch as anti-TGFβ antibodies, proteoglycans such as decorin and ligandsfor TGFβ receptors, and/or indirect TGFβ inhibitors includingaldosterone, inhibitors of aldosterone, inhibitors of angiotensin II,renin inhibitors, ACE inhibitors and AII receptor antagonists which actto decrease TGF production.

The methods of the invention also contemplate the use of agents thatresult in the enhanced degradation of excess accumulated matrixincluding proteases such as serine proteases including plasmin,metalloproteases, or protease combinations, and agents such as tPA, andPAI-1 mutants that increase the production and/or the activity ofproteases such as plasmin.

The agents for use in the methods of the invention may be administeredas inhibitory compounds in pharmaceutical formulations or as nucleicacid encoding the inhibitors delivered to suitable host cells. Thenucleic acid may be directly introduced into a cell in vivo, for exampleinto muscle tissue, or may be first introduced into a cell ex vivo toobtain a cell expressing the inhibitory agent or agents, and the cellthen transplanted or grafted into a subject to inhibit or reduce excessaccumulation of extracellular matrix.

The invention includes compositions for preventing or reducing theexcess accumulation of ECM containing a combination of agents forinhibiting TGFβ or a combination of agents for inhibiting TGFβ and fordegrading ECM.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting various pathways resulting in increasedTGFβ production.

FIG. 2 is a bar graph showing increases in TGFβ production by culturedhuman mesangial cells in response to renin, as described in Example I,infra,

FIG. 3 is a bar graph showing the effect of blocking agents onTGFβ-production by human mesangial cells in response to renin, asdescribed in Example II, infra.

FIGS. 4A and B are bar graphs showing dose dependent increases in TGFβ(FIG. 4A) and Fn production (FIG. 4B) with increases in HrRenin asdescribed in Example IV, infra.

FIGS. 5A and B are bar graphs showing time courses of TGFβ (FIG. 5A) andFn production (FIG. 5B) as described in Example IV, infra.

FIGS. 6A-C are bar graphs showing renin-induced increases in TGFβ, PAI-1and Fn mRNAs over time as described in Example IV, infra.

FIG. 7 is a bar graph showing the results of inhibitors that blockrenin's action to increase Angiotensin II, on the renin-induced increasein TGFβ production in adult human mesangial cells as described inExample IV, infra.

FIGS. 8A and B are photographs depicting the effects of tPA treatment onECM accumulation in glomeruli as described in Example V, infra.

FIGS. 9A-D are bar graphs depicting the effects of tPA treatment onamounts of ECM constituents (9A: FN EDA+; 9B: Laminin; 9C: Collagen Iand 9D: Collagen IV) as determined by staining as described in ExampleV, infra.

FIG. 10 is a bar graph showing the effects of tPA on glomerular mRNAexpression at day 6 as described in Example V, infra.

FIGS. 11A and B are bar graphs showing the effects of tPA treatment onglomerular plasmin activity as described in Example V, infra.

FIG. 12 is a bar graph demonstrating that injection of PAI-1 mutantresults in increases in plasmin generation of nephritic glomeruli, asdescribed in Example VII, infra.

FIG. 13 is a bar graph demonstrating decreased accumulation of Collagentype I after administration of PAI-1 mutant, as described in ExampleVII, infra.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery that a combination ofstrategies may be warranted to prevent or treat conditions associatedwith the excess accumulation of extracellular matrix in tissues ororgans, including fibrotic diseases and scarring resulting from TGFβoverproduction and/or activity. As previously reported, TGFβoverproduction may result from multiple pathways and require that morethan one pathway be inhibited to achieve any clinically significantreduction in excess accumulation of extracellular matrix andamelioration of disease. For example, as disclosed in co-pending U.S.patent application, U.S. Ser. No. 09/005,255, incorporated in itsentirety herein, renin stimulates TGFβ production in cells capable ofproducing TGFβ, in an angiotensin-II and blood pressure-independentmanner.

Optimal therapy of disorders associated with excess accumulation of ECMwhich causes organ impairment and ultimately failure, must take intoaccount the multiple pathways of TGFβ production to effectively combatoverproduction of TGFβ. Without such multifactorial stagy, inhibition ofone pathway of TGFβ production may be insufficient to block excessaccumulation of extracellular matrix and can even result in an increasein the levels of TGFβ production by stimulation of one of thealternative pathways for its production.

While it is now known that multiple stimuli result in TGFβoverexpression and resulting excess accumulation of ECM, therapeuticstrategies directly inhibiting TGFβ, such as the use of anti-TGFβantibodies or TGFβ receptor antagonists, are being explored. Howeverbecause TGFβ has many beneficial actions such as immunosuppressive andimmunomodulatory effects, as well as inhibition of epithelial cellgrowth which retards carcinogenesis (Markowitz, Science 268:1336-1338(1995) and suppression of atherogenesis (Grainger et al., Nature Med.1:74-79 (1995), these therapies may have unacceptable side-effects ifadministered at doses high enough to successfully stem fibroticconditions. This has been shown in the TGFβ 1 null (knockout) mice whichdie of overwhelming inflammation at about 6 weeks of age (Letterio etal., Science 264:1936-1938 (1994); Kulkami et al, Proc. Natl. Acad. Sci.USA 90:770-774 (1993) and Shull et al., Nature 359:693-699 (1992)),indicating that TGFβ1 has significant beneficial roles in immunefunction. Multiple agents, inhibiting TGFβ directly, and/or inhibitingthe disease specific stimuli underlying TGFβ overexpression and/oractivity, for example high glucose resulting from diabetes, may berequired to adequately reduce TGFβ-associated excess accumulation ofECM, without causing harmful side-effects. Accordingly, it is a goal ofthe methods of the present invention to accomplish normalization of TGFproduction without harmful side effects and to prevent or reduce excessaccumulation of ECM and ensuing fibrotic conditions.

In addition, degradation of accumulated ECM may be needed to restoretissue or organ function that has been compromised by the presence ofthe excess accumulated ECM. Prevention or degradation of excessaccumulated ECM can also prevent or reduce scar formation at the site ofa wound.

The methods of the invention include using multiple agents to reduce theoverproduction and/or activity of TGFβ and/or to block alternativepathways of TGFβ production to prevent or reduce excess accumulation ofECM. The methods of the invention further include the use of acombination of agents to reduce TGFβ overproduction and/or activity incombination with agents to enhance the degradation of excess,accumulated ECM. The methods are useful to prevent or reduce excessaccumulation of extracellular matrix to ameliorate fibrotic conditions,and to restore or maintain normal tissue or organ function or skinappearance.

As used herein “excess accumulation of extracellular matrix” means thedeposition of extracellular matrix components including, collagen,laminin, fibronectin and proteoglycans in tissue to an extent thatresults in impairment of tissue or organ function and ultimately, organfailure as a result of fibrotic disease. In addition, “excessaccumulation of extracellular matrix” means the deposition ofextracellular matrix components in the process commonly referred to as“scarring” or “scar formation,” e.g. at a wound site. “Reducing theexcess accumulation of extracellular matrix” means preventing excessaccumulation of extracellular matrix, e.g. in tissue, organs or at awound site, preventing further deposition of extracellular matrix and/ordecreasing the amount of excess accumulated matrix already present, tomaintain or restore tissue or organ function or appearance.

A variety of conditions are characterized by excess accumulation ofextracellular matrix (collagen, fibronectin and other matrixcomponents). Such conditions include, for example, but are not limitedto, glomerulonephritis, adult or acute respiratory distress syndrome(ARDS), diabetes-associated pathologies such as diabetic kidney disease,fibrotic diseases of the liver, lung and post infarction cardiacfibrosis. Also included are fibrocystic diseases such as fibrosclerosisand fibrotic cancers such as cancers of the breast, uterus, pancreas orcolon, and including fibroids, fibroma, fibroadenomas and fibrosarcomas.

There are also a number of medical conditions associated with an excessaccumulation of extracellular matrix involving increased collagen,fibronectin and other matrix components. Such conditions include, forexample, but are not limited to, post myocardial infarction, leftventricular hypertrophy, pulmonary fibrosis, liver cirrhosis,veno-occlusive disease, post-spinal cord injury, post-retinal andglaucoma surgery, post-angioplasty restenosis and renal interstitialfibrosis, arteriovenous graft failure, excessive scarring such as keloidscars and scars resulting from injury, burns or surgery.

As discussed, supra it is known that TGFβ is indicated in the causationof fibrotic conditions. During normal tissue repair, TGFβ production isincreased to stimulate the process of repair. When repair is complete,TGFβ production is reduced. If not reduced following normal tissuerepair, the increased TGFβ overproduction can result in the developmentof excess extracellular matrix accumulation and fibrotic conditions.Thus, repeated tissue injury or a defect in TGFβ regulation leading tosustained TGF production results in excess accumulation of extracellularmatrix.

As used herein “inhibition of TGFβ” includes inhibition of TGFβactivity, for example in causing excess deposition of ECM, as well asinhibition of TGFβ production resulting in overproduction and excessaccumulation of ECM, regardless of the mechanism of TGFβ activity oroverproduction. This inhibition can be caused directly, e.g. by bindingto TGFβ or its receptors, for example by anti-TGFβ antibodies or TGFβreceptor antagonists, or can be caused indirectly, for example byinhibiting a pathway that results in TGFβ production, such as the reninpathway. Inhibition causes a reduction in the ECM producing activity ofTGFβ regardless of the exact mechanism of inhibition.

As used herein a “TGFβ inhibitory agent” is an agent that directly orindirectly inhibits TGFβ binding to its receptors, such as aTGFβ-specific inhibitory agent, or an agent that blocks an alternativepathway of TGFβ production. The agent causes a reduction in the ECMproducing activity of TGFβ regardless of the mechanism of its action.The agent can be nucleic acid encoding the TGFβ inhibitory agent such asa cDNA, genomic DNA, or an RNA or DNA encoding TGFβ inhibitory activitysuch as a TGFβ antisense RNA or DNA.

As used herein, a “TGFβ-specific inhibitory agent” means an agentcontaining TGFβ inhibiting activity, including agents that bind directlyto TGFβ such as anti-TGFβ antibodies, or are a ligand for TGFβ whichprevents it from binding to its receptors. A TGFβ-specific inhibitingagent also includes a nucleic acid encoding a particular TGFβ-specificinhibitory agent such as a cDNA, genomic DNA or an RNA or DNA encodingTGFβ-specific inhibitory activity such as a TGFβ antisense RNA or DNA.

Agents that bind directly to TGFβ are known and include anti-TGFβantibodies such as anti-TGFβ1 antibodies (Genzyme, Cambridge, Mass.) andantibodies which bind both TGFβ1 and TGFβ2 (Dasch et al., U.S. Pat. No.5,571,714), proteoglycans such as decorin, biglycan and fibromodulin,and the nucleic acids encoding such agents.

Antibodies to inhibit TGFβ, renin or other molecules, for use in thepresent invention, can be prepared according to methods well establishedin the art, for example by immunization of suitable host animals withthe selected antigen, e.g. TGFβ. For descriptions of techniques forobtaining monoclonal antibodies see, e.g. the hybridoma technique ofKohler and Milstein (Nature 256:495-497 (1975)), the human B-cellhybridoma technique (Kosbor et al., Immunol. Today 4:72 (1983); Cole etal., Proc. Nat'l. Acad. Sci. USA, 80:2026-2030 (1983)) and theEBV-hybridoma technique (Cole et al., Monoclonal antibodies and CancerTherapy, Alan R. Liss, Inc., pp. 77096 (1985)). Such antibodies may beof any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclass thereof. The hybridoma producing the monoclonal antibody may becultivated in vitro or in vivo. Suitable host animals include, but arenot limited to, rabbits, mice, rats, and goats. Various adjuvants may beused to increase the immunological response to the host animal,depending on the host species, including, but not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as pluronic polyols, polyanions,peptides, oil emulsions, keyhole limpit, hemocyanin, dinitrophenol andpotentially useful human adjuvants such as BCG (Bacille Calmette-Guerin)and Cornebacterium parvum. Antibodies as used herein includes non-human,chimeric (different species), humanized (see Borrebaeck, AntibodyEngineering: A Practical Guide, W. H. Freeman and Co., New York, 1991),human and single-chain antibodies, as well as antibody fragmentsincluding but not limited to the F(ab′)₂ fragments that can be producedby pepsin digestion of antibody molecules and Fab fragments that can begenerated by reducing disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Science246:1275-1281 (1989)) to permit the rapid and easy identification ofmonoclonal Fab fragments having the desired specificity.

An indirect TGFβ inhibitor would inhibit the synthesis or secretion ofTGFβ or sequester it away from its target cells. Such inhibitorsinclude, but are not limited to, inhibitors of Angiotensin ConvertingEnzyme (“ACE”), antagonists of the AII receptor such as Losartan™ andCozar™ (Merck), and aldosterone inhibitors such as Spironolactone™(Sigma Chemical Co., St. Louis, Mo., Product # S 3378) that wouldotherwise result in increased TGFβ production.

Also included within the scope of TGFβ inhibitors of the invention arenucleic acids that include antisense oligonucleotides that block theexpression of specific genes within cells by binding a complementarymessenger RNA (mRNA) and preventing its translation (See review byWagner, Nature 372:332-335 (1994); and Crooke and Lebleu, AntisenseResearch and Applications, CRC Press, Boca Raton (1993)). Geneinhibition may be measured by determining the degradation of the targetRNA. Antisense DNA and RNA can be prepared by methods known in the artfor synthesis of RNA including chemical synthesis such as solid phasephosphoramidite chemical synthesis or in vitro and in vivo transcriptionof DNA sequences encoding the antisense RNA molecule. The DNA sequencesmay be incorporated into vectors with RNA polymerase promoters such asthe T7 or SP6 polymerase promoters. Alternatively, antisense cDNAconstructs that synthesize antisense RNA constitutively or inducibly canbe introduced into cell lines. The potency of antisense oligonucleotidesfor inhibiting TGFβ may be enhanced using various methods including 1)addition of polylysine (Leonetti et al., Bioconj. Biochem. 1:149-153(1990)); 2) encapsulation into antibody targeted liposomes (Leonetti etal., Proc. Natl. Acad. Sci. USA 87:2448-2451 (1990) and Zelphati et al.,Antisense Research and Development 3:323-338 (1993)); 3) nanoparticles(Rajaonarivony et al., J. Pharmaceutical Sciences 82:912-917 (1993) andHaensler and Szoka, Bioconj. Chem. 4:372-379 (1993)), 4) the use ofcationic acid liposomes (Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417 (1987); Capaccioli et al., Biochem. Biophys. Res. Commun.197:818-825 (1993); Boutorine and Kostina, Biochimie 75:35-41 (1993);Zhu et al., Science 261:209-211 (1993); Bennett et al., Molec. Pharmac.41:1023-1033 (1992) and Wagner, Science 280:1510-1513 (1993)); and 5)Sendai virus derived liposomes (Compagnon et al., Exper. Cell Res.200:333-338 (1992) and Morishita et al., Proc. Natl. Acad. Sci. USA90:8474-8478 (1993)), to deliver the oligonucleotides into cells. Recenttechniques for enhancing delivery include the conjugation of theantisense oligonucleotides to a fusogenic peptide, e.g. derived from aninfluenza hemagglutinin envelop protein (Bongartz et al., Nucleic AcidsRes. 22(22):4681-4688 (1994)).

Additional suitable TGFβ inhibitory agents can be readily obtained usingmethods known in the art to screen candidate agent molecules for bindingto TGFβ, such as assays for detecting the ability of a candidate agentto block binding of radiolabeled human TGFβ to cells such as humanmesangial cells. Alternatively, candidate compounds may be tested forthe ability to inhibit TGFβ production by mesangial cells using anenzyme-linked immunosorbent assay (ELISA), for example using the R & DSystems (Minneapolis, Minn.) TGFβ ELISA assay kit (Cat. No. DB 100) (formethods see, e.g. Uotila et al., J. Immunol. Methods 42:11 (1981)).

Suitable TGFβ-specific inhibitory agents can also be developed by knowndrug design methods, e.g. using structural analysis of the TGFβ moleculeemploying methods established in the art, for example, using X-raycrystallography to analyze the structure of the complex formed by TGFβand one of its known inhibitors (see, e.g. Sielecki et al., supra;Rahuel et al., supra, Badasso et al., supra and Dhanaraj et al.,supra.), and/or by modifying known TGFβ antagonists i.e. “leadcompounds,” to obtain more potent inhibitors and compounds for differentmodes of administration (i.e. oral vs. intravenous) (see, e.g. Wexler etal., Amer. J. Hyper. 5:209S-220S (1992)-development of AII receptorantagonists from Losartan™). For such procedures large quantities ofTGFβ can be generated using recombinant technology or purchasedcommercially (R & D Systems).

In addition to TGFβ inhibitory agents, agents that result in thedegradation of ECM are contemplated for use in the invention. Suchagents include serine proteases such as plasmin and metalloproteinases,and protease combinations such as Wobenzym (Mucos Pharma, Geretsried,Germany). In addition, the present inventors have discovered that agentssuch as tPA can be used to increase the amount of active proteases invivo to increase degradation of ECM accumulated in organs and tissues.Tissue plasmin activator (tPA, Activase, Genentech, S. San Francisco,Calif.) has been shown to dissolve clots associated with myocardialinfarction and stroke. The present inventors theorized that tPA might behelpful in increasing plasmin to reduce accumulated ECM. Shown herein isthe use of recombinant tPA (rtPA) to increase the generation of plasminin vivo to degrade ECM (Example V, infra).

In addition, new proteases or agonists of protease production and/oractivity may be discovered or developed using rational drug design andused to degrade ECM according to the methods of the present invention.

The present inventors have also discovered that PAI mutants, such as thePAI-1 mutants disclosed in WO 97/39028 by Lawrence et al., incorporatedby reference in its entirety herein, may be used to increase the amountof active plasmin to enhance degradation of ECM accumulated in organsand tissues. These PAI-1 mutants fail to inhibit plasminogen activators,yet retain significant vitronectin binding affinity. Additional PAI-1mutants for use in the methods of the invention may be obtained andtested for the ability to bind vitronectin while failing to inhibitplasminogen activators (Lawrence et al., J. Biol. Chem. 272:7676-7680(1997)). PAI-1 binding to vitronectin may be determined eitherfunctionally (Lawrence et al., J. Biol. Chem. 265:20293-20301 (1990)) orin a vitronectin specific ELISA (Lawrence et al., J. Biol. Chem.269:15223-15228 (1994)). The ability of PAI-1 to inhibit plasminogenactivators may be evaluated using chromogenic assays as described bySherman et al., J. Biol. Chem. 270:9301-9306 (1995)).

In the methods of the invention, the TGFβ inhibitory agents areadministered concurrently or sequentially. For example, an anti-TGFβantibody is administered with an anti-renin agent. The inhibitory agentswill localize at sites of TGFβ overproduction, e.g. organs such as thekidneys. The inhibitory agents may be labelled, using using knownradiolabelling methods to detect their localization in a subject afteradministration. The agents may also be conjugated to targeting moleculessuch as antibodies to ECM components to improve localization of theagents after administration to the sites of TGFβ overproduction and/orexcess accumulation of ECM in a subject.

In another embodiment of the methods of the invention, TGFβ inhibitoryagents are administered concurrently or sequentially with at least oneagent that degrades accumulated ECM, for example, a serine protease suchas plasmin. Alternatively, an agent that induces protease production,such as tPA, is administered to increase protease production at thesite(s) of accumulated ECM. tPA binds fibrin (Rondeau et al., ClinicalNephrol. 33:55-60 (1990)) and thus will localize in fibrotic areas wherethe increased protease production is desired.

In one embodiment of the invention, at least one TGFβ-inhibitory agentis administered to a subject having existing excess accumulation of ECMin tissues or organs, or at high risk for such accumulation to reduce orprevent excess accumulation of ECM. For example, individuals at risk fordeveloping fibrotic conditions, such as a person having or at high riskfor diabetes, high blood pressure, autoimmune disease (e.g. lupus) andinflammatory diseases can be scanned using known medical proceduresincluding tissue biopsies of kidney, lung or liver, to determine whetherECM has accumulated in these organs. If the agent is TGFβ-specific, itbinds to circulating TGFβ or tissue TGFβ. If the agent indirectlyinhibits TGFβ, for example an anti-renin agent, it reduces the amount ofTGFβ produced. As a result of the administration of agents that directlyor indirectly inhibits TGFβ, ECM that has accumulated at the time ofdiagnosis or treatment, as well as further accumulation of ECM isreduced. Moreover, in high risk individuals the methods of the inventionfor inhibiting TGFβ overproduction with multiple agents can result inprevention of excess accumulation of ECM and the development of fibroticconditions.

In another embodiment of the methods of the invention, at least one TGFβinhibitory agent is administered to a subject having an existing excessaccumuation of ECM in tissues or organs together with at least one agentto degrade accumulated ECM. The ECM degradation is accomplished using aprotease, or an agent that enhances production or the activity of ECMdegrading agents such as proteases. As a result of the administration ofthese agents, excess matrix accumulated at the time of diagnosis ortreatment, as well as further excess accumulation of ECM is reduced.

In addition to the use of molecules such as antibodies and purifiedcompound such as decorin, nucleic acid encoding the TGFβ inhibitoryagents and nucleic acid encoding the agent to directly or indirectlydegrade accumulated ECM, are administered to the subject to permit theagents to be expressed and secreted, for inhibiting TGFβ and degradingaccumulated ECM. The nucleic acid may be introduced into cells in thesubject, for example using a suitable delivery vehicle such as anexpression vector or encapsulation unit such as a liposome, or may beintroduced directly through the skin, for example in a DNA vaccine.

Alternatively, the nucleic acids encoding the agents are introduced intoa cell ex vivo and the cells expressing the nucleic acids are introducedinto a subject, e.g. by implantation procedures, to deliver the agentsin vivo. Multiple agents can be introduced into a delivery vehicle or inseparate vehicles.

Gene Therapy Methods

Methods for obtaining nucleic acids encoding TGFβ inhibitory agents andECM degrading agents are known in the art. Following is a generaldescription of methods of using the nucleic acids in gene therapy toreduce excess accumulation of ECM.

In one embodiment of the invention, gene therapy is contemplated usingnucleic acids encoding the TGFβ inhibitory agents and/or the ECMdegradation agent, introduced into cells in a subject to suppress TGFβoverproduction and to degrade accumulated ECM. Gene transfer into cellsof these nucleic acids is contemplated in the methods of the invention.

Nucleic Acids

Large amounts of the nucleic acid sequences encoding the TGFβ-inhibitingagents and/or the ECM degradation agents may be obtained usingwell-established procedures for molecular cloning and replication of thevector or plasmid carrying the sequences in a suitable host cell. DNAsequences encoding a specific agent can be assembled from cDNA fragmentsand oligonucleotide linkers, or from a series of oligonucleotides toprovide a synthetic inhibitor agent gene and/or ECM degradation genewhich can be expressed. Such sequences are preferably provided in anopen reading frame uninterrupted by internal non-translated sequences ofintrons, which are typically present in eukaryotic genes. Genomic DNAcontaining the relevant sequences can also be used. Sequences ofnon-translated DNA may be present 5′ to 3′ from the open reading frame,where such sequences do not interfere with manipulation or expression ofthe coding regions. Either complete gene sequences or partial sequencesencoding the desired agents are employed.

The nucleic acid sequences encoding the agents can also be produced inpart or in total by chemical synthesis, e.g. by the phosphoramiditemethod described by Beaucage and Carruthers, Tetra Letts. 22:1859-1862(1981) or the triester method (Matteucci et al., J. Am. Chem. Soc.103:3185 (1981) and may be performed on commercial automatedoligonucleotide synthesizers. A double-stranded fragment may be obtainedfrom the single-stranded product of chemical synthesis either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions, or by synthesizing the complementarystrand using DNA polymerase with an appropriate primer sequence.

Gene Transfer

For gene transfer, the key steps are 1) to select the mode of delivery,e.g. a proper vector for delivery of the inhibitor genes to the subject,2) administer the nucleic acid to the subject; and 3) achieveappropriate expression of the transferred gene for satisfactorydurations. Methods for gene transfer are known in the art. The methodsdescribed below are merely for purposes of illustration and are typicalof those that can be used to practice the invention. However, otherprocedures may also be employed, as is understood in the art. Most ofthe techniques to construct delivery vehicles such as vectors and thelike are widely practiced in the art, and most practitioners arefamiliar with the standard resource materials which describe specificconditions, reagents and procedures. The following paragraphs may serveas a guideline.

Techniques for nucleic acid manipulation are well known. (See, e.g.Annual Rev. of Biochem. 61:131-156 (1992)). Reagents useful in applyingsuch techniques, such as restriction enzymes and the like, are widelyknown in the art and commerically available from a number of vendors.

The natural or synthetic nucleic acid coding for the inhibitors forexpression in a subject may be incorporated into vectors capable ofintroduction into and replication in the subject. In general, nucleicacid encoding the selected inhibitor molecules and/or ECM degradationmolecules are inserted using standard recombinant techniques into avector containing appropriate transcription and translation controlsequences, including initiation sequences operably linked to the genesequence to result in expression of the recombinant genes in therecipient host cells. “Operably linked” means that the components are ina physical and functional relationship permitting them to function intheir intended manner.

For example, a promoter is operably linked to a coding sequence if thepromoter effects its transcription or expression.

Sequences encoding selected inhibitor and/or degradation genes willinclude at least a portion of the coding sequence sufficient to providethe TGFβ inhibitory or ECM degradation activity in the expressedmolecule. For example, in the case of a renin inhibitor, a portion ofthe coding sequence that enables the inhibitor to bind to renin can beused. Methods for determining such portions or “domains” includingbinding domains of molecules, are known in the art (See, e.g., Linsleyet al., Proc. Natl. Acad. Sci. USA 87:5031-5035 (1990)). It is possiblethat it may be necessary to block both the renin enzymatic site and therenin-cell binding domain in order to effectively prevent the stimulusto TGFβ overproduction by renin. In such case, renin antisense moleculescan be prepared using standard methods to accomplish complete blockade.

The selected nucleic acid sequences are inserted into a single vector orseparate vectors. More than one gene encoding a selected agent, orportion thereof containing the desired activity, may be inserted into asingle vector or into separate vectors for introduction into the hostcells. Alternatively, these sequences can be administered as nakednucleic acid sequences or as part of a complex with other molecules,e.g. liposomes.

A variety of expression vectors and gene transfer methods useful forobtaining expression of selected molecule in recipient cells are wellknown in the art, and can be constructed using standard ligation andrestriction techniques (see, for example, Sambrook et, al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989;Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y. (1982), Kriegler, Gene Transfer and Expression: ALaboratory Manual (W.H. Freeman and Co., New York, N.Y. 1990) and Wu,Methods in Enzymol. (Academic Press, New York, N.Y. 1993), each of whichis incorporated by reference herein). The choice of vector or methoddepends on several factors such as the particular molecule to beexpressed.

Suitable vectors may be plasmid or viral vectors (Kaufman, in GeneExpression Technology, Goeddel (Ed.) (1991)) including baculoviruses,adenoviruses, poxviruses (Moss, Current Opin. Biotech. 3:518-522(1993)), retrotransposon vectors (Cook et al., Bio/Technology9:748-751(1991) and Chakraborty et al., FASEB J. 7:971-977 (1993))adeno-associated viruses (AAV) (Yei et al., Gene Therapy 1:192-200(1994) and Smith et al., Nat. Genet. 5:397-402 (1993)), herpes virus andretrovirus vectors (Price et al., Proc. Natl. Acad. Sci. USA 84:156-160(1987); Naviaux and Verma, Current Opinion in Biotechnol. 3:540-547(1992); Hodgson and Chakraborty, Curr. Opin. Thera. Patients 3:223-235(1993)) such as the MMLV based replication incompetent vector pMV-7(Kirschmeier et al., DNA 7:219-225 (1988)), as well as human and yeastartificial chromosomes (HACs and YACs) (Huxley, Gene Therapy 1:7-12(1994) and Huxley et al., Bio/Technology 12:586-590 (1994)). Plasmidexpression vectors include plasmids including pBR322, pUC or Bluescript™(Stratagene, San Diego, Calif.).

Vectors containing the nucleic acid encoding the selected agents arepreferably recombinant expression vectors in which high levels of geneexpression may occur, and which contain appropriate regulatory sequencesfor transcription and translation of the inserted nucleic acid sequence.Regulatory sequences refer to those sequences normally associated (e.g.within 50 kb) of the coding region of a locus which affect theexpression of the gene (including transcription, translation, splicing,stability or the like, of the messenger RNA). A transcriptionalregulatory region encompasses all the elements necessary fortranscription, including the promoter sequence, enhancer sequence andtranscription factor binding sites. Regulatory sequences also include,inter alia, splice sites and polyadenylation sites. An internal ribosomeentry site (IRES) sequence may be placed between recombinant codingsequences to permit expression of more than one coding sequence with asingle promoter.

Transcriptional control regions include: the SV40 early promoter region,the cytomegalovirus (CMV) promoter (human CMV IE94 promoter region(Boshart et al., Cell 41:521-530 (1985)); the promoter contained in the3′ long terminal repeat of Rous Sarcoma Virus or other retroviruses; theherpes thymidine kinase promoter, the regulatory sequences of themethallothionein gene; regions from the human IL-2 gene (Fujita et al.,Cell 46:401-407(1986)); regions from the human IFN gene (Ciccarone etal., J. Immunol. 144:725-730 (1990); regions from the human IFN gene(Shoemaker et al., Proc. Natl. Acad. Sci USA 87:9650-9654 (1990);regions from the human IL-4 gene (Arai et al., J. Immunol. 142:274-282(1989)); regions from the human lymphotoxin gene (Nedwin et al., Nucl.Acids. Res. 13:6361-6373 (1985)); regions from the humangranulocyte-macrophage CSF gene (GM-CSF) (Miyatake et al., EMBO J.4:2561-2568 (1985)) and others. When viral vectors are used, recombinantcoding sequences may be positioned in the vector so that theirexpression is regulated by regulatory sequences such as promotersnaturally residing in the viral vector.

Operational elements for obtaining expression may include leadersequences, termination codons and other sequences needed or preferredfor the appropriate transcription and translation of the insertednucleic acid sequences. Secretion signals may also be included whetherfrom the native inhibitor or from other secreted polypeptides, whichpermit the molecule to enter cell membranes and attain a functionalconformation. It will be understood by one skilled in the art that thecorrection type and combination of expression control elements dependson the recipient host cells chosen to express the molecules ex vivo. Theexpression vector should contain additional elements needed for thetransfer and subsequent replication of the expression vector containingthe inserted nucleic acid sequences in the host cells. Examples of suchelements include, but are not limited to, origins of replication andselectable markers. Additionally, elements such as enhancer sequences,for example CMV enhancer sequences, may be used to increase the level oftherapeutic gene expression (Armelor. Proc. Natl. Acad. Sci. USA 70:2702(1973)).

The vector may contain at least one positive marker that enables theselection of cells carrying the inserted nucleic acids. The selectablemolecule may be a gene which, upon introduction into the host cell,expresses a dominant phenotype permitting positive selection of cellscarrying the gene ex vivo. Genes of this type are known in the art andinclude, for example, drug resistance genes such as hygromycin-Bphosphotransferase (hph) which confers resistance to the antibioticG418; the aminoglycoside phosphotransferase gene (neo or aph) from Tn5which codes for resistance to the antibiotic G418; the dihydrofolatereductase (DHRF) gene; the adenosine deaminase gene (ADA) and themulti-drug resistance (MDR) gene.

Recombinant viral vectors are introduced into host cells using standardtechniques. Infection techniques have been developed which userecombinant infectious virus particles for gene delivery into cells.Viral vectors used in this way include vectors derived from simian virus40 (SV40; Karlsson et al., Proc. Natl. Acad. Sci. USA 82:158 (1985));adenoviruses (Karlsson et al., EMBO J. 5:2377 (1986)); vaccinia virus(Moss et al., Vaccine 6:161 (1988)); and retroviruses (Coffin, in Weisset al. (Eds.), RNA Tumor Viruses, 2nd Ed., Vol. 2, Cold SpringLaboratory, NY, pp. 17-71 (1985)).

Nonreplicating viral vectors can be produced in packaging cell lineswhich produce virus particles which are infectious but replicationdefective, rendering them useful vectors for introduction of nucleicacid into a cell lacking complementary genetic information enablingencapsidation (Mann et al., Cell 33:153 (1983); Miller and Buttimore,Mol. Cell. Biol. 6:2895 (PA317, ATCC CRL9078). Packaging cell lineswhich contain amphotrophic packaging genes able to transduce cells ofhuman and other species origin are preferred.

Vectors containing the inserted inhibitor genes or coding sequences areintroduced into host cell using standard methods of transfectionincluding electroporation, liposomal preparations, Ca-PH-DNA gels,DEAE-dextran, nucleic acid particle “guns” and other suitable methods.

In additional to various vectors including viral vectors, other deliverysystems may be used including, but not limited to, microinjection(DePamphilis et al., BioTechnique 6:662-680 (1988)); liposomal mediatedtransfection (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417(1987); Felgner and Holm, Focus 11:21-25 (1989) and Felgner et al.,Proc. West. Pharmacol. Soc. 32:115-121 (1989)); use of naked or particlemediated DNA transfer and other methods known in the art. Recently,cationic liposomes have been used to enhance transfection (Felgner etal., Nature 349:351 (1991); Zhu et al., Science 261:209 (1993)).

Suitable host cells for gene transfer consist of vertebrate cells suchas fibroblasts, keratinocytes, muscle cells, mesangial cells (see,Kitamura et al., Kidney Int. 48:1747-1757 (1995)), and any othersuitable host cell including so-called universal host cells, i.e. cellsobtained from a different donor than the recipient subject butgenetically modified to inhibit rejection by the subject. Autologouscells are preferred, but heterologous cells are encompassed within thescope of the invention.

Expression of the selected TGFβ inhibitor genes after introduction intothe host cells is confirmed using standard methods. For example,expression of TGFβ-specific inhibitory agents can be determined byassaying for the ability of the supernatant from transfected cells toinhibit the binding of radiolabeled TGFβ to human mesangial cells usingFluorescent Activated Cell Sorting (FACS) or ELISA. Expression from hostcells of an agent that inhibits TGFβ indirectly, such as Losartan, canbe confirmed by detecting a decrease in fibronectin production bymesangial cells exposed to supernatant from transfected cells, relativeto controls. Expression of genes encoding ECM degrading agents can bedetermined using, for example, an in vitro system using mesangial cellscultured on a ECM substrate such as Matrigel™ (Collaborative Research,Inc., Bedford, Mass.) that contains the major components of themesangial matrix, including laminin, type IV collagen, entactin andheparan sulfate proteoglycan, as described by Baricos et al., KidneyInternatl. 47:1039-1047 (1995)). The ECM substrate is radiolabeled andECM degradation by the product of an expressed gene from transfectedhost cells is determined by measuring the release of radioactivity fromthe ECM into serum-free medium. These assay systems may also be employedto screen candidate TGFβ inhibiting and ECM degrading agents.

Administration of TGFβ Inhibitory Agents and Agents DegradingAccumulated ECM

Agents for inhibiting TGFβ and agents for degrading accumulated ECM aresuspended in physiologically compatible pharmaceutical carriers, such asphysiological saline, phosphate-buffered saline, or the like to formphysiologically acceptable aqueous pharmaceutical compositions foradministration to a subject. Parenteral vehicles include sodium chloridesolution, Ringer's desctrose, dextrose and sodium chloride and lactatedRinger's solution. Other substances may be added a desired, such asantimicrobials.

The TGFβ inhibiting and ECM degrading agents may be administeredtogether or apart, simultaneously or sequentially, to carry out themethods of the invention.

Modes of administration of the TGFβ inhibitory agents and ECM degradingagents are those known in the art for therapeutic agents and includeparenteral, for example, intravenous (e.g. for antibody inhibitors orproteases), intraperitoneal, intramuscular, intradermal, and epidermalincluding subcutaneous and intradermal, oral (e.g. small molecule reninand TGFβ antagonists), or applied to mucosal surfaces, e.g. byintranasal administration using inhalation of aerosol suspensions, andby implanting to muscle or other tissue in the subject (e.g. for genetransfer of nucleic acid expressing renin and/or TGFα inhibitors).Suppositories and topical preparations are also contemplated.

The TGFβ inhibitory and ECM degrading agents are introduced in amountssufficient to prevent or reduce excess accumulation of extracellularmatrix in susceptible tissues and organs including, but not limited to,lung and kidney tissue. Before or after administration, if necessary toprevent or inhibit the subject's immune response to the vehiclescarrying the inhibitors, immunosuppressant agents may be used.Alternatively, the vehicles carrying the TGFβ inhibitory and ECMdegrading agents can be encapsulated.

The most effective mode of administration and dosage regimen for theTGFβ inhibitory and ECM degrading agents for use in the methods of thepresent invention depend on the extent of TGFβ overproduction, theseverity of the accumulation of extracellular matrix and resultingimpairment of tissue or organ function, the subject's health, previousmedical history, age, weight, height, sex and response to treatment andthe judgment of the treating physician. Therefore, the amount of TGFβinhibitory and ECM degrading agents to be administered, as well as thenumber and timing of subsequent administrations, are determined, by amedical professional conducting therapy based on the response of theindividual subject. Initially, such parameters are readily determined byskilled practitioners using appropriate testing in animal models forsafety and efficacy, and in human subjects during clinical trials ofcandidate therapeutic formulations. Suitable animal models of humanfibrotic conditions are known (see, e.g. Border and Noble, New Eng. J.Med. 331:1286-1292 (1994), incorporated by reference herein).

After administration, the efficacy of the therapy using the methods ofthe invention is assessed by various methods including biopsy of kidney,lung or liver or other tissue to detect the amount of extracellularmatrix accumulated. An absence of significant excess accumulation ofECM, or a decrease in the amount or expansion of ECM in the tissue ororgan will indicate the desired therapeutic response in the subject.Preferably, a non-invasive procedure is used to detect a therapeuticresponse. For example, changes in TGFβ activity can be measured inplasma samples taken before and after treatment with an inhibitor (see,Eltayeb et al., J. Am. Soc. Nephrol. 8:110A (1997)), and biopsy tissuecan be used to individually isolate diseased glomeruli which are thenused for RNA isolation. mRNA transcripts for TGFβ, and extracellularmatrix components (e.g. collagen) are then determined using reversetranscriptase-polymerase chain reaction (RT-PCR) (Peten et al., J. Exp.Med. 176:1571-1576(1992)).

Advantages of the Invention

The invention provides improved treatment and prevention of fibroticconditions associated with overproduction of TGFβ and excessaccumulation of ECM in tissues and/or organs resulting in impairedfunction, or scarring, by reducing TGFβ overproduction directly and thatresulting from multiple biological pathways, to effectively inhibit theTGFβ induced component of extracellular matrix deposition, and byincreased degradation of ECM using degrading agents.

The therapeutic effects of the invention result from a reduction in orprevention of the TGFβ-induced excess accumulation of extracellularmatrix in tissues and/or organs, and when combined with ECM degradingagents, from the increased degradation of ECM over time.

The following examples are presented to demonstrate the methods of thepresent invention and to assist one of ordinary skill in using the same.The examples are not intended in any way to otherwise limit the scope ofthe disclosure of the protection granted by Letters Patent grantedhereon.

EXAMPLE I Demonstration that Renin Upregulates TGFβ in Human MesangialCells

Normal fetal human mesangial cells (Clonetics Corp., Clonetics,Walkersville, Md.) passaged 5 to 8 times, were plated (3,000 cell/cm) in12 well plates in 2 ml of medium (Mesangial Basal Medium (CloneticsCorp.) containing 5% FCS, 10 μg/ml penicillin and 100 μg/mlstreptomycin) and allowed to grow to confluence for 48 hours at 37° C.,5% CO₂. Cultures were washed three times using sterile phosphatebuffered saline at room temperature and then 2 ml/well of serum free MBMmedium to induce quiescence. After 48 hours, the serum-free medium wasremoved and 2 ml/well of fresh serum-free medium was added. Humanrecombinant renin (Hoffman-La Roche Ltd., Basel, Switzerland) inconcentrations from 10⁻⁶ to 10⁻¹²M was added to each well. A blank and 5ng/ml of TGFβ (R & D Systems, Minneapolis, Minn.) were used as controls.Cells and supernatants were harvested by centrifugation after 24 hrs ofculture and frozen at 10° C. until analysis. The total production andrelease of TGFβ into the culture supernatant was measured using an ELISAkit (R & D Systems). Induction of PAI-1 and fibronectin in thesupernatant are also measured using anti-PAI-1 and anti-fibronectinantibodies in an ELISA to provide further confirmation of the inhibitionof TGFβ. TGFβ, fibronectin and PAI-1 mRNA are measured usingsemi-quantitative RT-PCR.

(1) Determination of Dose Dependence of Renin Induction of TGFβ

As shown in FIG. 2, renin increases the TGFβ production by culturedhuman mesangial cells in a dose-dependent manner.

EXAMPLE II DEMONSTRATION OF THE EFFECT OF INHIBITING RENIN ON TGFβPRODUCTION BY HUMAN MESANGIAL CELLS

Renin inhibitor Ro42-5892 (Hoffman-LaRoche, Basel, Switzerland),Losartan™ (Merck Pharmaceuticals, West Point, Pa.), Enalapril™ (SigmaChemical Co., St. Louis, Mo., Prod. No. E6888), or TGFβ1 neutralizingantibody (R & D Systems) were added in the amounts indicated below toseparate wells in triplicate to block the renin cascade at differentsites after stimulation by renin:

-   10⁻⁵M Renin Inhibitor Ro 42-5892 (Hoffman-LaRoche)-   30 ng/ml Anti-TGFβ1 antibody (R & D Systems, #AB 101 NA)-   30 ng/ml Chicken IgG (control for anti-TGFβ1 antibody, R & D    Systems, # AB 101 C)-   10⁻⁵M Enalapril™ (Sigma Chemical Co., St. Louis, Mo.)-   10⁻⁵M Losartan (Merck Pharmaceuticals, West Point, Pa.)

These inhibitors were added at zero time with 10⁻⁷M human recombinantrenin (Hoffman-LaRoche).

As shown in FIG. 3, use of inhibitors that block renin's action toincrease Angiotensin II i.e. blocking Angiotensin I production fromAngiotensinogen (Ro 42-5892), blocking Angiotensin I conversion toAngiotensin II (Enalapril™) and blocking binding of Angiotensin II toits type 1 receptor (Losartan™), does not reduce the renin-inducedincrease in TGFβ production. These results demonstrate for the firsttime an alternative pathway in which TGFβ production is stimulated byrenin.

EXAMPLE III Demonstration of Inhibition of TGFβ by Blocking Renin InVivo in the Presence of an Anti-Fibrotic Drug

In this example, a known fibrotic disease drug, Enalapril™ whichinhibits the production of Angiotensin II, is combined with an inhibitorof renin, antisense renin oligonucleotide, to obtain an enhancedtherapeutic effect on fibrotic disease in an animal model.

Rats are administered Enalapril™ in their drinking water prior toanti-thymocyte serum injection, e.g. three (3) days prior to injection.Anti-thymocyte antibody, e.g. OX-7, is injected intravenously into therats at day three to produce fibrotic disease. (Bagchus et al., Lab.Invest. 55:680-687 (1986)). Renin antisense oligonucleotides areadministered one hour following administration of OX-7 by introducingthe oligonucleotides into a suitable vehicle, such as HVJ liposomes, andinjecting the formulations into the left renal artery of Sprague Dawleyrats as described for renin genes by Arai et al., Biochem. And Biophys.Res. Comm. 206(2):525-532 (1995), incorporated by reference herein. Acontrol consisting of nonsense encoding oligonucleotides (e.g. derivedfrom the renin antisense gene sequence) is also injected into the leftrenal artery of additional rats. The renin antisense localizes in thejuxtaglomerular apparatus of the glomerulus where renin is producedblocking renin production.

Animals are sacrificed on day 7 and kidney tissue samples are taken foranalysis of levels of TGFα in the glomeruli. Glomeruli are sievedindividually from each rat and placed in culture in suitable medium forthree days. At the end of culture, culture supernatant, is harvested bycentrifugation and TGFβ, fibronectin and PAI-1 production are determinedas makers of fibrotic renal disease severity. Other glomeruli are pooledand used to isolate RNA. RNA is used by standard methods to quantitateexpression of mRNAs of interest, including TGFβ, fibronectin andcollagens.

Glomeruli are also examined histologically for phenotypical changes,e.g. changes resulting from deposition for ECM. Phenotypic changes areassociated with pathological alteration of glomeruli indicative offibrotic disease. Such changes include expansion of extracellular matrixin the mesangial area of the kidney in animal models and the presence ofactivated mesangial cells which have acquired the characteristics offibroblasts, e.g. expressing α-smooth muscle actin and interstitialcollagen, indicating progressive glomerular injury (Johnson et al., J.Am. Soc. Nephrol. 2:S190-S197 (1992)). Tissue for light microscopy isfixed in formaldehyde, then dehydrated in graded ethanol and embedded inparaffin. Sections are cut at 3 μm thickness and are stained with withthe periodic Schiff reagent. The paraformaldehyde-fixed renal section ofthe rats are also incubated with mouse anti-human renin monoclonalantibody (Kaiichi Radioisotope Labs, Ltd., Tokyo, Japan), mouseanti-α-smooth muscle actin monoclonal antibody (Immunotech S. A.(Marseille, France) and rabbit anti-collagen antibodies (Chemicon,Temicula, Calif., prod. No. AB755). The sections are further processedusing Vectastain ABC Kit (Vector Laboratories, Inc., Burlingame,Calif.).

Results of antibody binding indicate the extent of glomerular injury andthe effects of inhibition of renin on such injury.

EXAMPLE IV Additional Demonstration that Renin Upregulates TGFβ in HumanMesanglil Cells

Primary cultures of adult human mesangial cells were grown from humannephrectomy tissues using standard methods. Cells were passaged 4-7times and then plated (3,000 cell/cm2) in 12 well plates in 2 ml ofmedium (Mesangial Basal Medium (Clonetics Corp, containing 5% FCS, 10μg/ml penicillin and 100 μg/ml streptomycin) and allowed to grow to 70%confluency for 48 hours at 37° C., 5% CO₂. Cultures were washed threetimes using sterile phosphate buffered saline at room temperature andthen 2 ml/well of serum free MBM medium to induce quiescence. After 48hours, the serum-free medium was removed and 2 ml/well of freshserum-free medium was added for 24 hours. Human recombinant renin(HrRenin, Hoffman-La Roche Ltd., Basel, Switzerland) in concentrationsfrom 10⁻⁶ to 10⁻¹²M was added to each well for 24 hours. A blank (noHrRenin) was used as a control. Cells and supernatants were harvested bycentrifugation after 24 hrs of culture and frozen at 70° C. untilanalysis.

The total production and release of TGFβ into the culture supernatantwas measured using an ELISA kit (R & D Systems). Induction of the matrixprotein fibronectin (Fn) in the supernatant was measured usinganti-fibronectin antibodies in an ELISA to provide further confirmationof induction of TGFβ. Renin-induced induction of TGFβ, fibronectin andPAI-1 mRNA were measured over time using semi-quantitative RT-PCR in amultiple# system where multiple cDNAs are amplified simultaneouslyaccording to Dostal et al., Anal. Biochem. 223:239-250 (1994),incorporated by reference herein Determinations were done in triplicatemesangial cell cultures.

(1) Determination of Dose Dependency of Renin Induction of TGFβ

As shown in FIG. 4, statistically significant (p<0.05) dose dependentincreases in TGFβ (FIG. 4A) and Fn production (FIG. 4B) were observed,peaking with 2- and 1.4-fold increases at 10⁻⁶M HrRenin, respectively.Time course experiments using 10⁻⁷M HrRenin revealed significantincreases in TGFβ and Fn production at 24 and 48 hours (p<0.03 (FIGS. 5Aand B). As shown in FIGS. 6A-C, renin-induced increases in TGFβ, PAI-1and Fn mRNAs peaked at 4 hours with increases from 1.5- to 2-fold.

(2) Demonstration that Renin Upregulation of TGFβ is not MediatedThrough Renin Enzymatic Activity or Angiotensin II

Renin inhibitor Ro42-5892 (Hoffman-LaRoche, Basel, Switzerland),Losartan™ (Merck Pharmaceuticals, West Point, Pa.), Enalapril™ (SigmaChemical Co., St. Louis, Mo., Prod. No. E6888), or TGFβ1 neutralizingantibody (R & D Systems) were added in the amounts indicated below toseparate wells in triplicate to block the renin cascade at differentsites after stimulation by renin:

-   10⁻⁵M Renin Inhibitor R042-5892 (Hoffman-LaRoche)-   10⁻⁵M Enalapril™ (Sigma Chemical Co., St. Louis, Mo.)-   10⁻⁵M Losartan (Merck Pharmaceuticals, West Point, Pa.)-   Controls included neutralizing antibody to TGFβ (ATG) and control    IgG (TgG)

These inhibitors were added at zero time with 10⁻⁷M human recombinantrenin (Hoffman-LaRoche).

As shown in FIG. 7, use of inhibitors that block renin's action toincrease Angiotensin II, i.e. blocking Angiotensin I production fromAngiotensinogen (RO 42-5892), blocking Angiotensin I conversion toAngiotensin II (Enalapril™) and blocking binding of Angiotensin II toits type I receptor (Losartan™), does not reduce the renin-inducedincrease in TGFβ production.

These results provide additional evidence that renin upregulates TGFβproduction by human mesangial cells through a mechanism which isindependent of renin's enzymatic action to convert angiotensin toAngiotensin I, and independent of Angiotensin II generation. Theseresults may have profound implications for progression of fibrotic renaldisease, particularly in states of high plasma renin as are observedwith therapeutic Angiotensin II blockade. Thus, the use of therapeuticagents such as Enalapril™ or Losartan™ for Angiotensin blockade may notbe optimal as treatment agents because of resulting high renin levels,preventing a therapeutic reduction in TGFβ. In addition, antagonistsdeveloped to block the site on renin that acts in the Angiotensin IIpathway, would not be expected to block the action of renin that isindependent of this pathway. Therefore, effective therapy of fibroticdiseases must take these multiple pathways for TGFβ increase intoconsideration.

EXAMPLE V Demonstration of the Ability of tPA to Increase PlasminDegradation of Accumulated ECM In Vivo

In this Example, recombinant tissue type plasminogen activator (rtPA)was shown to promote generation of the protease plasmin in nephriticglomeruli and to degrade pathological ECM proteins leading to atherapeutic reduction in matrix accumulation.

Six Sprague-Dawley rats with were injected with phosphate bufferedsaline (PBS, as a control) and 18 rats were injected with 300 ug ofmouse monoclonal OX7 antibody produced in the laboratory usingcommercially obtained hybridoma cells (American Type Culture Collecton(Rockville, Md., USA; Peters et al., Kidney Internatl. 54:1570-1580(1998)) on day 1 to induce anti-Thy-1 nephritis. Injection of the antirat-thymocyte antibody intravenously causes binding to an epitope in ratglomerular mesangial cells call Thy 1.1. The complement-mediatedmesangial cell lysis that follows initiates a cascade of tissue injury,followed by a repair process that involves induction of TGFβ-drivenproduction and deposition of ECM components. In addition, the plasminprotease system is altered such that PA is decreased and PAI-1 ismarkedly increased. These alterations favor decreased plasmin generationwhich decreases matrix turnover and enhances matrix accumulation.Plasmin is the key to mesangial cell matrix turnover (Baricos et al,Kidney Int. 47:1037-1047 (1995)).

Three days after the initial injection, rtPA (Genentech, Inc., SanFrancisco, Calif.) in a formulation designed for rodent intravenousinjection (GenBank E08757) or PBS was injected intravenously. Injectionswere repeated twice a day from day 3 to day 5. RtPA was injected i.v. ata dose of 1 mg/kg BW (n=6). Controls received saline (n=6). Glomerularstaining for ECM matrix proteins (collagen type I and III, fibronectionEDA+ and tenascin) and glomerular mRNA levels of TGFβ1, fibronectin andPAI-1 were evaluated at day 6. Localization of rtPA in nephriticglomeruli and the effect of rtPA on glomerular plasmin wereinvestigated. Rats were sacrificed at day 6 and kidney tissues excised,fixed in formalin and frozen for histological analysis.

TABLE 1 Groups of Six Rats Treatment Group 1 - Normal controls 300 ug ofPBS on day 1, then 300 ug PBS 2X Group 2 - Disease control 300 ug of OX7on day 1, then 300 ug PBS 2X Group 3 - Disease + Dose 1 300 ug of OX7 onday 1, then 0.25 mg/day rtPA 2X/day on days 3, 4 and 5

Kidney tissue sections were stained for extracellular matrix usingPeriodic Acid Schiff (PAS) using standard procedures and were stainedfor specific relevant matrix proteins such as Collagen 1, Collagen IV,Fibronectin EDA and tenascin using standard immunohistochemical stainingprocedures, Matrix proteins were scored by image analysis of 30glomeruli per rat.

FIGS. 8A (control) and B (tPA) show an overall decrease in matrixaccumulated as a result of tPA treatment. Compared to the untreated,disease control group (FIGS. 9A-D), the percentage of the glomerulararea with positive staining was significantly lower in the rtPA treatedgroup at day 6 for fibronectin EDA+(FN)(19±2 vs. 14±1, p<0.01), laminin(35±2 vs. 25±2, p<0.001), type I collagen 33±1 vs. 21±3, p<0.001) andtype IV collagen (27±2 vs. 23 ±1, p<0.01). Glomerular levels of TGFβ1,FN and PAI-1 mRNA were unchanged (FIG. 10). rtPA co-localized withfibrin along the glomerular capillary loops and in the mesangium.

rtPA was injected into nephritic rats 10, 20 and 30 minutes beforesacrifice. At sacrifice, glomeruli were isolated and placed in culturewith a chromogenic substrate for tPA. Plasmin generation by nephriticglomeruli, as shown in FIG. 11, was significantly elevated in tPAtreated nephritic glomeruli compared to nephritic gomeruli from diseasecontrol rats.

This example demonstrates that injected rtPA binds fibrin in nephriticglomeruli where it increases plasmin generation and promotespathological ECM degradation. rtPA may thus be used in the methods ofthe invention as an ECM degrading agent.

EXAMPLE VI Effect of Administration of TGFβ Inhibitory Agents and Agentsthat Promote Degradation of ECM

In this example, at least one agent that inhibits TGFβ, anti-TGFβantibody or decorin, is administered in combination with an ECMdegrading agent, such as rtPA to reduce excess ECM accumulation anddegrade accumulated ECM in an animal model of glomerulonephritis.

Sprague-Dawley rats are treated as described in the above Examples toinduce nephritis. Groups of six (6) rats each include untreated diseasecontrols, rats treated with tPA alone as in Example V, above, ratstreated with Enalapril™ alone (200 mg/day) in drinking water and ratstreated with both intravenous rtPA and Enalapril™ in drinking water. Onday 6 rats are sacrificed and kidney sections are excised, fixed informalin and frozen for histological analysis. Glomeruli are isolatedand used for in vitro analysis of production of TGFβ, fibronectin andPAI-1 using ELISA assays of culture supernatants and for isolation ofRNA for Northern analysis of message levels of TGFβ, fibronectin andPAI-1. Tissue samples are stained for ECM proteins and glomerular mRNAlevels of TGFβ1, fibronectin and PAI-1.

It is expected that the results of treatments with both anti-TGFβantibody and rtPA treatment are significantly lower positive stainingboth in PAS stained tissue and in glomeruli stained for specific matrixcomponents, as shown in Example V, compared with groups treated witheither agent alone or in the control disease group.

EXAMPLE VII Demonstration of the Effects of Administration of a PAI-1Mutant on Extracellular Matrix Degradation

The human PAI-1 mutant used in this experiment (see WO 97/39028); wasconstructed on the wild-type PAI-1 background (Ginsburg et al., J. Clin.Invest 78:1673-1180 (1986)), and disabled by the introduction of two Argresidues at positions 333 and 335 of the mature protein, which are alsoreferred to as residues P14 and P12 of the reactive center loop(Lawrence, Adv. Exp. Med. Biol. 425:99-108 (1997)). Upon interactionwith a proteinase, these substitutions greatly retard the insertion ofthe reactive center loop into β-sheet A and prevent the mutant fromadopting the latent conformation. Since loop insertion results in lossof vitronectin affinity (Lawrence et al., 1997, supra), the PAI-1 mutantretains significant vitronectin activity while failing to inhibit allplasminogen activators.

Four to six week old male Sprague-Dawley rats (Sasco, Inc., Omaha,Nebr.) were treated as described in the above Examples to induceanti-thy-1 nephritis by intravenous injection of the monoclonalanti-thymocyte antibody OX-7 350 mg/200 g body weight. Groups of six (6)rats included a normal control group (injected with saline), anuntreated disease control group (injected with PBS), and a group treatedwith 1 mg/Kg PAI-1 mutant injected once a day beginning 24 hours afterinduction of ATS nephritis and ending at day 5. Two additional groups ofrats were treated with 1) 100 mg/liter of Enalapril (in drinking water)with a loading dose of Enalapril given by gavage 24 hr after diseaseinduction followed by 100 mg/liter of Enalapril in drinking water, and2) a 6% low protein diet (Teklad, Madison, Wis., diet number TD86551)started 24 hours following disease induction.

Rats were sacrificed at day 6 and kidney tissues excised, fixed informalin and frozen for histological analysis. Kidneys were perfused insitu with cold buffered saline (PBS) at pH 7.4, and then excised. Piecesof cortex were removed and either snap frozen in 2-methylbutane that hadbeen cooled in liquid nitrogen or fixed in 10% neutralized formalin forimmunohistologic examination. The capsules were removed and the corticaltissue dissected out and minced with a razor blade prior to isolation ofglomemli by standard graded seiving. Kidney tissue sections were stainedfor extracellular matrix using Periodic Acid Schiff(PAS) using standardprocedures and were stained for specific relevant matrix proteins suchas Collagen I, Collagen IV, Fibronectin EDA and tenascin using standardimmunohistochemical staining procedures. Matrix proteins were scored bya blinded observer. 20 glomeruli per rat were evaluated. Isolatedglomeruli were also used to determine glomerular mRNA levels of TGFβ1,fibronectin and PAI-1 at day 6.

Reagents to measure plasmin activity, including plasminogen, lowmolecular weight u-PA and H-D-Val-Leu-Lys-p-nitroanilide (S-2251) wereobtained from KabiVitrum (Franklin, Ohio). PAI-1 activity was assayed bymeasuring the hydrolysis of synthetic substrate by formed plasmin in thepresence of plasminogen (Marshall et al., J. Biol. Chem. 265:9198-8204(1990)). Assays were performed in polyvinyl chloride microtiter plates.The total volume of 125 μl was comprised of the following: sample,H-D-Val-Leu-Lys-P-nitroanilide (0.01 μM) and plasminogen (0.03 μM) in0.5% Triton X-100, 0.1M Tris, at pH 8.0. The amount of p-nitroanilinereleased was measured at 410 nm with a Thermomax microplate reader(Molecular Devices, Menlo Park, Calif.). A standard curve was generatedwith each assay using low molecular weight human u-PA. Each sample wasalso assayed without plasminogen to establish the plasminogen dependenceof the enzyme activity. The plasmin activity in culture supernatant orcell lysate was expressed as IU/1000 glomeruli.

FIG. 12 shows an increase in plasmin generation of glomeruli in cultureas a result of injection of the PAI-1 mutant. Compared to the untreated,disease control group, the glomerular plasmin activity was significantlyhigher in the PAI-1 treated group, being approximately halfway betweenthe activity of disease controls and normal glomeruli. Notably, thesignificant increase in glomerular plasmin activity in nephriticglomeruli was observed with the PAI-1 mutant 24 hours following thefinal injection.

In addition, treatment with the PAI-1 mutant resulted in decreasedaccumulation of Collagen Type I, relative to diseases controls (FIG.13), while glomerular levels of TGFβ1, FN, PAI-1 mRNA and Collagen ImRNA were not significantly altered. The decreased accumulation ofCollagen Type I together with the fact that the Collagen I mRNA does notsignificantly decrease suggests enhanced extracellular matrixdegradation rather than decreased production of Collagen I.

These results suggest that the increase in glomerular plasmin activitywith a PAI-1 mutant can be titrated to avoid large increases in plasmingeneration that may lead to hemorrhaging. Thus, the dose of the PAI-1mutant may be altered, for example by doubling the dose, increaseglomerular plasmin activity to normal, but not excessive, levels todecrease deleterious accumulation of extracellular matrix. In addition,the time of treatment may be extended, for example to 10 days to obtaindesired degradation.

Various publications are cited herein that are hereby incorporated byreference in their entirety.

As will be apparent to those skilled in the art in which the inventionis addressed, the present invention may be embodied in forms other thanthose specifically disclosed without departing from the spirit orpotential characteristics of the invention. Particular embodiments ofthe present invention described above are therefore to be considered inall respects as illustrative and not restrictive. The scope of theinvention is as set forth in the appended claims and equivalents thereofrather than being limited to the examples contained in the foregoingdescription.

1. A method for treating a condition associated with the excessaccumulation of extracellular matrix in a tissue and/or organ or at adermal wound site comprising: a) reducing the excess accumulation ofextracellular matrix associated with TGFβ overproduction and/or activityin an organ or tissue, or at a wound site, by administering in an amountsufficient to inhibit TGFβ overproduction and/or activity, a first agentthat reduces TGFβ-associated accumulation of extracellular matrix; andb) degrading excess accumulated extracellular matrix in said tissueand/or organ or wound site, by administering in an amount sufficient todegrade excess accumulated extracellular matrix, a different, secondagent that degrades extracellular matrix; whereby the accumulation ofextracellular matrix in said tissue and/or organ or wound site isreduced from the level existing at the time of treatment.
 2. The methodof claim 1 wherein the accumulation of extracellular matrix is reducedto a level which does not interfere with normal functioning of thetissue or organ or result in scarring.
 3. The method of claim 1 whereinsaid first agent that reduces TGFβ-associated accumulation ofextracellular matrix is selected from the group consisting of inhibitorsof aldosterone, inhibitors of angiotensin II, anti-TGFβ antibodies,renin, ACE inhibitors, AII receptor antagonists, proteoglycans andligands for the TGFβ receptor.
 4. The method of claim 3 wherein said ACEinhibitor is Enalapril™.
 5. The method of claim 1 wherein said step ofdegrading excess accumulated extracellular matrix comprises contactingsaid matrix with at least one protease in an amount sufficient todegrade excess accumulated extracellular matrix to a level that does notimpair the normal function of said tissue and/or organ or result inscarring.
 6. The method of claim 5 wherein said at least one protease isselected from the group consisting of serine proteases,metalloproteinases and protease combinations.
 7. The method of claim 1wherein said step of degrading accumulated extracellular matrixcomprises administering an agent which increases the amount of activeprotease sufficient to degrade excess accumulated matrix to a level thatdoes not impair the normal function of said tissue and/or organ orresult in scarring.
 8. The method of claim 7 wherein said protease isselected from the group consisting of serine proteases,metalloproteinases and protease combinations.
 9. The method of claim 8wherein said protease is plasmin and said agent which increases theamount of active plasmin is tPA.
 10. The method of claim 1 wherein saidcondition associated with the excess accumulation of extracellularmatrix is a fibrotic condition.
 11. The method of claim 10 wherein saidfibrotic condition is selected from the group consisting ofglomerulonephritis, adult or acute respiratory distress syndrome (ARDS),diabetes, diabetic kidney disease, liver fibrosis, kidney fibrosis, lungfibrosis, post infarction cardiac fibrosis, fibrocystic diseases,fibrotic cancer, post myocardial infarction, left ventricularhypertrophy, pulmonary fibrosis, liver cirrhosis, veno-occlusivedisease, post-spinal cord injury, post-retinal and glaucoma surgery,post-angioplasty restenosis, renal interstitial fibrosis, arteriovenousgraft failure and scarring.
 12. The method of claim 1 wherein saidtissue or organ is selected from the group consisting of kidney, lung,liver, heart, arteries, skin and the central nervous system.
 13. Themethod of claim 1 wherein said condition associated with the excessaccumulation of extracellular matrix is scarring.
 14. The method ofclaim 1 wherein said first agent that reduces TGFβ-associatedaccumulation of extracellular matrix is nucleic acid encoding the agent.15. The method of claim 5 wherein said protease is nucleic acid encodinga protease.
 16. The method of claim 1, wherein said first agent thatreduces TGFβ-associated accumulation of extracellular matrix and saidsecond agent that degrades excess accumulated extracellular matrix areadministered concurrently.
 17. The method of claim 1 wherein said firstagent that reduces TGFβ-associated accumulation of extracellular matrixand said second agent that degrades excess accumulated extracellularmatrix are administered sequentially.
 18. A method for treating acondition associated with the excess accumulation of extracellularmatrix in a tissue and/or organ, or at a wound site, comprisingadministering in an amount sufficient to inhibit TGFβ activity and/orproduction, a combination of at least one first agent and at least onesecond agent, to prevent or reduce the excess accumulation ofextracellular matrix associated with TGFβ overproduction in a tissueand/or organ, or at a wound site, said first agent an agent that reducesTGFβ-associated accumulation of extracellular matrix, and said secondagent an agent that degrades extracellular matrix.
 19. The method ofclaim 18 wherein said agents are nucleic acids encoding said at leastone first agent and at least one second agent.
 20. The method of claim18 wherein said agents are administered to reduce TGFβ overproductionprior to excess accumulation of extracellular matrix.